PROJECT SUMMARY Measuring interactions of molecules with membrane proteins on cells and quantifying the interaction kinetics in real time are critical for understanding many cellular processes, for validating biomarkers, and for screening drugs. This is because membrane proteins are responsible for many important cellular functions of cells, including communication with other cells, sensing the surrounding environment, and transporting molecules and ions in and out of cells. They also comprise nearly 60% of current drug targets. However, developing such a capability has been a difficult challenge, especially for small molecules, because most traditional binding kinetics measurement technologies are based on the detection of molecular mass, which diminishes with the size of the molecule. Small molecules are the most important forms of drugs, accounting for over 70% of all the drugs developed to date. To address the unmet need, this project will develop a mechanically amplified optical detection technology. The technology is based on a basic thermodynamics principle that a mechanical deformation in the cell membrane occurs when a molecular binding event takes place on the cell. By accurately monitoring the mechanical deformation, one can thus determine the kinetics of both large and small molecule binding with membrane proteins on cells. This new strategy provides mechanical amplification to small binding signals, which, together with a novel imaging technology and signal processing algorithm to track cell deformation with sub-nanometer accuracy, make it possible to detect molecular interactions with membrane proteins. It also allows the study of heterogeneous nature of cells by analyzing the binding kinetics variability between different cells. Finally, the technology allows the study of membrane proteins that are difficult or impossible to isolate from cells with intact native structures and activities. The specific aims of the project are to 1) establish data acquisition and analysis algorithms to accurately detect molecular binding-induced cell membrane deformation, 2) develop a high-throughput system for single cell binding kinetics analysis, 3) develop a low-noise imaging technology for studying low-density membrane proteins, and 4) validate the performance and usability of the new technology. The success of the project will lead to a new tool for biomedical research community and industry for studying basic cellular processes, for validating biomarkers, and for screening new drugs.